Indian Journal of Experimental Biology Vol. 40, September 2002, pp. 1026-1031

Comparative immunopotentiating properties of saponin and incomplete Freund's adjuvant coupled to affinity purified larval antigen of

Hyalomma anatolicum anatolicum

S Ghosh*, N K Singh & Preeti Rawat

Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Iza tnagar 243 122, India

Received 24 Jalluary 2002; revised 9 July 2002

Comparati ve immunostimulatory properties of saponin and incomplete Freund's adjuvant (/FA) were studied using af­finity purified 39 kDa larval antigen of Hyalolllma oll(l/oliCUIIl ollalOlicul1l. Significant antibody response to 39 k a antigen was detected in the sera of rabbits immunized with both 39 kDa plus saponin and 39 kDa plus IFA in comparison to thei r corresponding control animals. Insignifican t differences were noted in the antibody response between the animal s of two immunized groups. Upon challenge, the animals immunized with 39 kDa plus IFA rejected 76.2±9.7% of larvae and 4S.S±4. 1 % of adu lts while in group of animals injected with 39 kDa plus sapon in rejected SO.9± 11.2% of larvae and 47 .2 ±S.7% of adults.

For immunization, adjuvant is mixed with an antigen to enhance the immunogenicity to levels beyond that produced by the antigen alone. A good adjuvant should be non-toxic, non-pyrogenic! and must in­crease the immunogenicity of a candidate protein by many-folds without producing adverse reactions at the site of inj ection2

. These are some of the important considerations in developing immunoprophylactic measures against the diseases of livestock and the se­lection of a suitable adjuvant is an essential aspect for the development of any effective vaccine.

Ticks and tick-borne di seases are major contraints to raise a viable li vestock industry, particularly in de­veloping countries like India. In recent years, increas­ing problem of acaricidal resistance to ticks3 has stimulated research on development of anti-tick vac­cines. Significant progress has been made in develop­ing vaccine against Boophilus microplus and recom­binant vaccines have been released4

.5

. However, in India, a few research groups are working on the de­velopment of vaccines against Hyalomma anatoliculI1 anatolicum, vector of bovine tropical theileriosis . Re­cently, a group of antigens have been isolated and tested for their immunoprotec tive potential s against experimen tal challenge infestations6

.8

. To immunise cross-bred ani mals all the purified antigens were mixed with FCA or IFA and immune responses were correlated with the level of protective immunity.

*Correspondent aut hor - Fax: 058 J -440368 ; E-mai l : sghosh@ivri .up.n ic.in

Aim of the present study was to inves tigate the comparative effects of saponin and IFA on the chal­lenge infestation of rabbits to 39 kDa purified larval antigen of Hyalomma anatolicum anarolicum to de­velop better delivery system.

Materials and Methods Animals-New Zealand white rabbits (n=16) of 3-

4 months of age were procured from the laboratory animal research division of the Institute and were maintained on standard diet (150 g of green fodder per animal per day) in disinfected cages (70 x 70 x60 cm).

Antigen-Antigen extraction was done in an ice bath at 4°C and extracts were stored at -20°C. Larval antigen of H.a. anatolieum was prepared as described by Ghosh and Khan9

. The larval homoge nate (TLE) was chromatographically purified as described by Ghosh et al. 6

• Briefly, immunoglobulins were precipi­tated from two animals immunized with soluble larval antigen of H.a. anatolieum and protected against chal­lenge infestations9 as per the method of Fey et al.!o. IgG was further isolated from the total immunoglobu­lins!! and was coupled to CNBr activated Sepharose 4B and an immunoaffinity column was generated as per the recommendation of Pharmaci" Fine Chemi­cals. TLE was charged on the affini ty column at a flow rate of 15 ml/hr. and the bound antigen was eluted with 0.1 M, glycine-HCl , pH 2.8, into Iris-base to increase pH to 7.2. Eluted antigen was dialysed extensively against equilibrating buffer, concentrated

GHOSH el 01.: IMMUNOPOTENTIATING PROPERTIES OF SAPONIN & H. ANA TOLICUM ANATOLICUM 1027

with polyethylene glycol 20,000 and designated as Aff-TLE. Protein content of the antigen was estimated by spectrophotometric method 12.

Polyacrylamide gel electrophoresis -Eluted bound antigen was electrophoretica ll y separated on I mm thick gel using a discontinuous systeml3. Stack­ing gel was 3% acrylamide in 0.5 M Tris, pH 6.8 with 0.25 % of SDS. Gels were stained with Coomassie brilliant blue R-250 to identi fy the marker proteins which ranged from 97.4 to 29 kDa and puri fied ti ck protein .

Experimen.tal design-Two adjuvants, saponin and IFA were tested for their ab ility to potentiate an­tibody response to 39 kDa purified larval antigen. The animals of group A (n=4) were immunized with 200 , 200 and 100 Ilg of 39 kDa an ti gen emulsified with IFA and inj ected on weeks I, 2 and 4, respectively. For immunization of animals of group B (n=4), 500 Ilg of ant igen was suspended with saponin ( I mg mr ') and injected simultaneously with the animals of group A. Two separate groups (n=4,C and D) of rab­bits were maintained as control and received compa­rable amount of phosphate buffer (pH 7.3) plus either IFA or sapon in . All the rabb its were challenged on 10Ih day of last immuni zat ion with laboratory reared disease-free 500 larvae and 10 pairs of H. a. anatoli­cum ad ults. The ear bags were checked daily and the feed ing period of various stages was calculated as :

Number of fed larvae/ad ults dropped --------------------~--xIOO

Number of larvae/adults released

The percentage rejecti on was calcul ated as 100 -percentage recovery.

Engorgement weight of dropped larvae and adu lts were recorded. Dropped ad ults were kept individually at 30o ± 2°C and 80±5% RH . The pre-oviposition period, ovi position period and egg massess were re­corded. The engorged larvae were kept at the same temperature and humidity and moulting period and moulting percentage was recorded. Moulting percent­age was calcul ated as:

Number of subsequent stage emerging

M I· 01 after moulting

ou tlng 10 = -------=--------- -Total number of larvae feeding to engorgemen

Stmistica/ ana/ysis- Di fference between means were determined using Student' s t test l4.

EnZYll1e linked inl/I/ull osorbellf assay (ELlSA) ­Blood samples from all the an i mals were coll ected every ten days from 0 to 60 days post-immunization

(DPI) . To study the antibody responses, polystyrene microtitre plates (Greiner, Frickenhansen, Germany) were coated with Aff-TLE diluted in carbonate­bicarbonate buffer (pH 9.6) . The antigen was diluted so th at each well contai ned 1 Ilg of protei n. The plates were left overni ght at 4°C 15 and then washed four times with PBS (pH 7.2) containing 0.05% tween-20. After four washes, the wells were blocked by BSA (1 %) in PBS (pH 7.2) for 2 hr. After washing, 100 III of experimental or control sera (1: 1000) were added to each well and incubated for 2 hr. The pl ates were again washed 4 times and 100 III of affinity purified anti-rabbit IgG conjugated with HRPO (S igma Chemical Company, St. Louis, MO, USA) was added to each well and incubated fo r 2 hr. After four washes with washing buffer, 100 III of orthophenylenedi a­mine in phosphate citrate buffer (pH 5.0) was added. The plates were finally kept in the dark for 10 min and the reacti on was stopped by addi ng SO III of O. IN H2S04 in each well. OD values were measured at 492 nm using a multi scan plate reader.

Results Affinity chroll/atography and SDS-PAGE-A total

of I SO mg total larval extracts was loaded in batches and 2.8 mg bound protein was eluted (Fig. I ). SDS­PAGE revealed a single band of 39 kDa in the eluent (Figs 2,3).

Antibody response - Rabbits immuni zed with Aff­TLE plus IFA (group A) gave significant ant ibody response in compari son to corresponding control an i­mals as earl y as on 20 DPI. Increas ing trend was con­tinued upto 60 DPI. Mean OD values obtained in the sera of animals collected on different days was 0.05, 0.97, 1.97,2.6,3.4,3.75 and 3.7 times hi gher than the corresponding control an imals and most of the va lues were signifi cant at P < 0.0 I level.

1.6

1.4

1.2

E 1 C

'" '" ..,. 0.6 -;;; g 06

0.4

0.2 0.1 M Glycine He l pH 2.8

\ 1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 16

Fraction numbers

Fig. I --Showing elution pallern o/" AIT - TLE.

1028 IND IAN J EXP BIOL, SEPTEMBER 2002

As in the case of animals of group A, a steady in­crease of anti body response was also noted in the an imals of group B. Mean 00 values obtained in the sera of animals co ll ected on different days was found 0,0.6, 1.3,2.0,2.9,3.3 and 3.4 times higher than the corresponding control an imals and the data were sig­nificant at P <O.OI level. There was no signifi cant difference between antibody level st imulated by in-

A B c

.,

M

kDo 974

68

43

Fig . 2 - SDS-PAGE profile or TLE (lane C) unbound (lane A), ArI'-TLE (lane B) and marker pro te ins (lane M).

Fig . 3-39 kDa protein used ror immuni za ti on.

k Do 39

oculat ion of 39 kOa prote in plus l FA (group A) and 39 kOa protein plus saponin (group B; F ig. 4) .

Feeding and de velopillen t pattem or lar\'oe ­Effect of immuniza ti on on the larvae is shown in Table I . Immed iate hypersens iti vity reaction s were not observed at the feeding sites . Larvae were seen dying within 48 hI' of allachment in thc immunized rabbits and consequentl y a sign if icantly higher per­cen tage of larvae were rejected from the an imals of groups A and B in compari son to their co rresponding control group. Among the dropped larvae collec ted f rom the animals of all the groups, a signifi cant ly lesser percentage of larvae fed on immun ized groups ( 14.9% in group A , 12.4% in group 13) mou lted to nymphs in compari son to the larvae fed on animals of corresponding cont ro l group. Immuni za tion of ani­mals also resulted in a signifi cant reduction in the nymphal population in group A and in group B in compari son to their corresponding control groups. When immunostimulati ve properties or IFA and saponin

0.7

0.6

E 0.5 c

N 0.4 en "<t

iU 0.3

0 a 0.2

0.1

o

I= ~=~I _D

,.'

10 20 30

Days

40 50 60

Fi g. 4 - Antibody response or rabbit s followin g imm Llniz:lti o n:

Gr A - inocu lated wi th 39 kDa protei n + irA; and Gr

B - inocu lated w ith 39 kDa protein + sapon in anc! Gr C and D se rved as con tro l.

Table 1- Feed in g and deve lopment pe rrormances of larvae of H a. alla/o /ic lIIlI fed on rabbits immu­nized wi th AfI'-TLE plus IFA, AfI'-TLE pl us saponin and unimmuni zed cont ro l

IVa lues a rc ex pressed as mea n ± SE 01'4 replica tions l

Group

A B

C D

Rejec tion (%)

76.2 ± 9.7 b

80.9± ll.i'

28.5 ± 12.2

21.7±8.2

Feeding period (days)

5.6± 0.3

4.8±0.2

5.2± 0.4

4.9 ± 0.3

S igniriean t at P: "<0.001 ; h<O.O I ; '<0.05

Mo ulting Moulting (%) period

(days)

14.9±4.2" 13.0±0.4

12.4±3. 1" 13.2±0.2

52.1 ± 5.4 10.2±0.4

58.7±3.9 11.4 ±0.6

Overall decrease

in resultant nymphs (%)

96.4±9.7'

97.6 ±7 .Sh

62 .7±8.4

54.0±7.2

Group A , rabbits immuni zed wit h AfI'-TLE plus IFA; group B, imillu nized w ith Arf-TLE plus saponin; groups C and D, unimmuni zed control rabbits whi ch rece ived injection or phosphate bufrer pl us e ithe r IFA or sa ponin

GHOSH ('/ 01.: IMMUNOPOTENTIAT IN G PROPERTIES OF SAPON IN & H ANIlTOLlCUM IINATOLICUM 1029

in combination wi th Aff-TLE was compared , it was observed that although there was a slight differences in the feedin g and developmental performances of larvae fed on animals of groups A and B, the differ­ence was not stat istically signi ficant. No signifi cant difference was noted in the feed ing and moulting pe­riod of larvae fed on immunized and control rabbits.

Feeding and reproductive pelformances of adullS­

Table 2 summari ses the comparative immunoprotec­tive properties of IFA and saponin in combinati on with Aff-TLE on the feeding and reproductive per­fo rmances of adults fed on immunized and co nt ro l anima ls. Hi gh an tibody levels in an imals of both the immuni zed groups interfere in adu lt biology as ev i­denced in chall enge, when a signifi cantly hi gher per­centage of adul ts (45.3 %, in group A and 47 .2%; in group B) was rejected from the immuni zed anima ls. The adult ti cks fed on animals of groups A and B were li ghter (162 and 122.3 mg, respecti ve l y) than the ticks fed on groups C and 0 and the mean differences in weight were found hi ghl y sign ificant. Although, the egg masses laid by the ticks fed on immuni zed an i­mal s were signi ficantl y less in comparison to the ticks fed on control an imals, the reproductive index was signifi cantly lower in the ticks fed on animal s of group A on ly. When feed in g and reproductive per­forma nce of adults fed on animal s of groups A and B were co mpared it was observed that the mean en­gorged weight of adu lts fed on group A animal s was lesser (24.6 mg) than the ti cks fed on group B anima ls but the difference was not stati stically signifi cant. However, a significant difference was noted in the egg mass laid by the ti cks fed on groups A and B ani­mals . Insignificant differcnces were observed in the engorgement , pre-ovipositi on and ov ipos iti on periods of ticks fed on imlllunized and contro l rabbits .

Discussion In the broader sense an immunolog ica l adjuvant

can be described as a substance that acts nonspecifi­ca ll y to enhance the response to an ant igen. Mineral

·1 h . f· . 16 TI 0 1 S are t e maInstay o · veteri nary vacc ll1es. ley are genera ll y poor at promoting a ce ll medi ated re­sponse l 7

. A good humoral and cellular immune re­sponses are essential for successful co nt ro l of para­sitic and viral diseases l8

. The best known adju vant combinat ion is Freund's complete adjuvant (FCA) whi ch combines the immunomod ul atory properti es of Mycobacterium tuberculosis alongwith the short-term depot effect of water in oil emu lsions . However, FCA can cause chronic innammation and induce auto il1l -

I· . 19 mune comp Icatlons .

I n the present study, although both the preparations provided comparative protective immunity aga inst larvae and adults of H. a. anatoliculII , the reproduct ive index of ticks fed on group A an imals was Illuch lesser than the ticks fed on group B animal s. In contras t, sapon in has been shown superi or to IFA when combined to gut membrane antigen in co nferring protection against B. l71icropLus challenge infestations20

. Opdebeeck et al. 2 1 have used IFA with soluble gut ant igen and quil A with gut membrane anti gen of B. microp/us and 91 % protection against larva l cha llenge (3x20000) was noted in the an imals immunized wi th gut membrane anti gen plus quil A, whi le insign ificant protection has been recorded in the an imals immunized with gut soluble antigen pl us IFA . An ex trapolation of the work of Bomford21

, who has demonstrated that the adjuvant of choice for the so luble antigen is IFA, and a particulate antigen is saponin, Ghosh el a16

. immunized cross-bred calves with 39 kDa purified anti gen of H. a. alla/oliculll mixed with IFA and a signifi cant protection against larvae (7 1.6%) and nymphs (77.3%) has been noted.

Table 2 - Colllparative feeding and reproductive performances of adull S o f H. a. ({l/olUliclIIl/ fed on rabbits immun ized with An·-TLE plus IFA , AIT-TLE plu s saponin and lInimlllunized control

I Values arc exp ressed as mea n ± SE of 4 replications]

Groups Rejec lion Engorgc ment (% ) period (days)

A 45.8± 4.1 " 8.9±0.4

B -17.2 ± 5.7" 8.7±0.2

C 31.6±4.8 91 ±0.3

D 28.7± 5.9 8.8 ± 0.2

Significant at- F: "<G.OO I: b<O.O I : "<0 .05 A, 13, C , D, as indicated in Table I

Engorged Pre-ov iposition O vipos iti on w I. (mg) period (clays) period (days)

3 17.8± 12.6" 11.7±0.6 17.9±0.6

342.0 ± 1.9" 12.2±0.4 16.2±0.2

479.0 ± 10.0 12.4±0.6 18.8±0.1

464.0 ± 10.9 12.6 ± 0.2 19. 1 ±0.3

Egg Reproductive masses index

157.2 ± 8.7" 0.49±0.01 "

183.4±7.8" 0.53 ±0.02

274 .0±5.7 0.57±0.01

264.4±6.8 0.56±0.02

1030 INDI AN J EXP BIOL, SEPTEMB ER 2002

However, Wong and Opdebeeck22 have shown that both so luble and particul ate fo rm of gut membrane antigens of B. microplus can evoke signifi cant protecti ve immuni ty against chall enge in fes tations when combined with quil A. Similarly, Smith el al. 23

have induced signi ficant protec ti on in cattl e against Babesia bovis using soluble anti gens with qui! A. Their results suggest that the soluble or parti cul ate nature of the antigen is not the only consideration in the choice of suitable adju vant fo r induction of immunological response and protective imm unity.

In the case of H a. analo/iculIl, FCA has been used ex tensively for experi mental immun ization of rabbi ts and cross-bred calves and signi ficant protection against challenge in festa tions has been reported9.24.25. Later, FCA has been replaced by IFA for immuniza­tion of animals against experi mental chall enge infes­tation and recorded protection against larvae (7 1.6%) and nymphs (77.3 %)6. Recentl y, FCA/IFA has been used with purified anti gens for immunizati on of cross­bred animals against experimental challenge in fes ta­tions and achi eved signi ficant protecti on 7.8.

Saponin has the special properties of surfactant ad­juvant as has been descri bed by Hun ter el a/.26 and Hunter and Bennet27

, but the immunopotenti ating properties of sapon in has not been tested earli er with the pu ri fi ed anti gens of H a. analoliclIlIl. The present finding suggested that the vesicle adj uvant, IFA, could be re placed by the surfactant adju va nt, sapo nin , for immuni zati on of cross-bred cattle against in festa­tion of H a allalo/icul1I.

Acknowledgement Sincere thanks are due to the Director, IVRI fo r

prov iding laboratory fac ili ties. The author is grateful to Dr S C Gupta, Principal Sc ienti st, Di vision of Para­sitology, IVRI , Izatnagar fo r technical help .

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